a) Field of the Invention
The invention is directed to a method in fluorescence microscopy, particularly laser scanning microscopy, for analyzing predominantly biological samples, preparations and associated components. Included are methods based on fluorescence detection, e.g., fluorescence correlation microscopy, methods for screening active ingredients (high throughput screening), total internal reflection fluorescence microscopy (TIRF), and nearfield scanning microscopy.
The transition from the detection of a few broad-spectrum dye bands to the simultaneous acquisition of whole spectra opens up new possibilities for the identification, separation and correlation of mostly analytic or functional sample characteristics. These include spectral, spatial and dynamic characteristics or combinations of these characteristics. Simultaneous analyses of many types of markings of chromosomes (multicolor banding (MCB) and FISH or related techniques) can be carried out in metaphase and interphase samples for scientific or diagnostic purposes.
Through the simultaneous detection of complete spectra, the detection efficiency is substantially increased compared to prior art methods. The resulting increase in sensitivity makes it possible to detect smaller dye concentrations and/or reduced intensity of the excitation light that it used. In this way, the bleaching effects and toxic effects of excitation can be minimized.
b) Description of the Related Art
A typical area of application of light microscopy for examining biological preparations is fluorescence microscopy (Pawley, “Handbook of Biological Confocal Microscopy”; Plenum Press 1995). In this case, determined dyes are used for specific labeling of cell parts.
The irradiated photons having a determined energy excite the dye molecules, through the absorption of a photon, from the ground state to an excited state. This excitation is usually referred to as single-photon absorption (FIG. 1a). The dye molecules excited in this way can return to the ground state in various ways. In fluorescence microscopy, the most important transition is by emission of a fluorescence photon. Because of the Stokes shift, there is generally a red shift in the wavelength of the emitted photon in comparison to the excitation radiation; that is, it has a greater wavelength. Stokes shift makes it possible to separate the fluorescence radiation from the excitation radiation.
The fluorescent light is split off from the excitation radiation by suitable dichroic beam splitters in combination with blocking filters and is observed separately. This makes it possible to show individual cell parts that are dyed with different dyes. In principle, however, multiple parts of a preparation can also be dyed simultaneously with different dyes which bind in a specific manner (multiple fluorescence). Special dichroic beam splitters are used again to distinguish the fluorescence signals emitted by the individual dyes.
In addition to excitation of dye molecules with a high-energy photon (single-photon absorption), excitation with a plurality of lower-energy photons is also possible (FIG. 1b). The sum of energies of the single photons corresponds approximately to a multiple of the high-energy photon. This type of excitation of dyes is known as multiphoton absorption (Corle, Kino, “Confocal Scanning, Optical Microscopy and Related Imaging Systems”; Academic Press 1996). However, the dye emission is not influenced by this type of excitation, i.e., the emission spectrum undergoes a negative Stokes shift in multiphoton absorption; that is, it has a smaller wavelength compared to the excitation radiation. The separation of the excitation radiation from the emission radiation is carried out in the same way as in single-photon excitation.
The prior art will be described in the following by way of example with reference to a confocal laser scanning microscope (LSM) (FIG. 2).
An LSM is essentially composed of four modules: light source, scan module, detection unit and microscope. These modules are described more fully in the following. In addition, reference is had to DE19702753A1.
Lasers with different wavelengths are used in an LSM for specific excitation of different dyes in a preparation. The choice of excitation wavelength is governed by the absorption characteristics of the dyes to be examined. The excitation radiation is generated in the light source module. Various lasers (argon, argon-krypton, helium-neon, solid state lasers, diode lasers, Ti:Sa lasers, etc.) are used for this purpose. Further, the selection of wavelengths and the adjustment of the intensity of the required excitation wavelength is carried out in the light source module, e.g., using an acousto-optic crystal. The laser radiation subsequently reaches the scan module via a fiber or a suitable mirror arrangement.
The laser radiation generated in the light source is focused in the sample in a diffraction-limited manner by the objective through the scanner, scan optics and tube lens. The scanner scans the sample in a point raster in x-y direction. The pixel dwell times when scanning over the sample are mostly in the range of less than one microsecond to several seconds.
In confocal detection (descanned detection) of fluorescent light, the light emitted from the focal plane (specimen) and from the planes located above and below the latter reaches a dichroic beam splitter (MDB) via the scanner. This dichroic beam splitter separates the fluorescent light from the excitation light. The fluorescent light is subsequently focused on a diaphragm (confocal diaphragm/pinhole) located precisely in a plane conjugate to the focal plane. In this way, fluorescent light components that were generated outside of the focus are suppressed. The optical resolution of the microscope can be adjusted by varying the diameter of the diaphragm. Another dichroic blocking filter (emission filter EF) which again suppresses the excitation radiation is located behind the diaphragm. After passing the blocking filter, the fluorescent light is measured by means of a point detector (PMT).
When using multiphoton absorption, the excitation of the dye fluorescence is carried out in a small volume in which the excitation intensity is particularly high. This area is only negligibly larger than the detected area when using a confocal arrangement. Accordingly, a confocal diaphragm can be dispensed with and detection can be carried out directly following the objective (nondescanned detection).
In another arrangement for detecting a dye fluorescence excited by multiphoton absorption, descanned detection is carried out again, but this time the pupil of the objective is imaged in the detection unit (descanned detection).
In a sample extending in three dimensions, only the plane of the sample (optical section) located in the focal plane of the objective is reproduced by the two detection arrangements in connection with corresponding single-photon absorption or multiphoton absorption. By recording a plurality of optical sections in the x-y plane at different depths z of the sample, a three-dimensional image of the sample can be generated subsequently in a computer-assisted manner.
Accordingly, the LSM is suitable for examination of thick preparations. Different fluorescence dyes are characterized by different absorption spectra and emission spectra. The laser lines for exciting the dye or dyes are selected in accordance with the absorption spectra. Dichroic filters adapted to the emission characteristics of the dye or dyes ensure that only the fluorescent light emitted by the respective dye will be measured by the point detector.
Currently, in biomedical applications, a plurality of different cell regions are labeled simultaneously by different dyes (multifluorescence). In the prior art, the individual dyes can be detected separately based either on different absorption characteristics or on emission characteristics (spectra) (FIG. 3a). FIG. 3a shows the emission spectra of different typical dyes. The intensity of the emission is plotted as a function of wavelength. It will be noted that the dyes designated by 1 to 4 differ with respect to the position and shape of their emission spectra.
According to the prior art, existing methods known in the art for spectral splitting of emission signals can be divided into two categories:
A Sequential Data Acquisition:
    1) Combination of a spectrally dispersive element with monochromatic detection    2) Methods of interferometric spectroscopy    3) Multitracking, i.e., change in excitation wavelength according to image or line recording for separating dyes with different absorption characteristics (reference to our application)B Parallel Data Acquisition:    1) Spectral splitting of the fluorescence emission by means of secondary color splitting and emission filtration.
Methods A1 and B3 are applied in the LSM 510 laser scanning microscope by Zeiss.
Flow cytometers are used for analyzing and classifying cells and other particles. For this purpose, the samples are dissolved or suspended in a liquid and are pumped through a capillary. In order to examine the samples, a laser beam is focused in the capillary from the side. The samples are labeled with different dyes or fluorescing biomolecules. The fluorescence emission and the scattered excitation light are measured. The art is described in “Flow Cytometry and Sorting”, second edition, M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds., Wiley & Sons, Inc., New York, 1990, pp 81-107.
The size of the samples can be determined from the scattered signal. Different sample particles can be separated and/or sorted or counted separately by means of the spectral characteristics of the fluorescence of individual samples. The sorting of the sample particles is carried out with an electrostatic field in different collecting vessels. The evaluation of this technique is carried out by means of histograms which provide information about the intensities of the labeling and about the quantity of differently labeled samples. The through-flow rate is typically about 10-100 cm/s. Therefore, a highly sensitive detection is necessary. According to the prior art, a confocal detection is carried out in order to limit the detection volume.
The accuracy of the through-flow measurement is influenced by different factors. Such factors are, for example, nonspecific fluorescence, autofluorescence of cells, fluorescence of optical components and the noise of the detectors that are used.
Disadvantages of the Methods of the Prior Art
Dyes with sharply overlapping excitation and emission spectra can hardly be separated from one another without crosstalk. This problem becomes more severe as the quantity of fluorescence dyes to be detected increases. Therefore, a unique correlation of the emission of a dye to a detection channel is impossible. Yet this is absolutely necessary for accurate assessment in the analysis of multiply labeled samples.
Other troublesome and unwanted effects in multifluorescence recordings are superimposed background signals. These background signals can be reflections of individual lasers at the sample or broadband autofluorescence signals of sample constituents that overlap the spectral signatures of the fluorescence-labeled sample locations to be analyzed and therefore make it difficult and sometimes even impossible to analyze these sample locations.
Chromosomes that are labeled with up to seven different dyes by means of multicolor banding technique, FISH or related techniques pose special demands for the detection and separation of the individual markings. Samples of this kind can be analyzed according to the prior art by all of the methods listed above under A and B. These methods have the following disadvantages:
In B1, the spectral splitting of fluorescence emissions by means of secondary color splitting and emission filtration, the emission spectra intersect increasingly as the quantity of dyes increases. This results in crosstalk. Accordingly, it is impossible to uniquely correlate the emission of a dye to a detection channel.
Method A3 (multitracking) only solves the problem when the excitation spectra are sufficiently different from one another. However, this is not the case when a plurality of dyes are used.
Methods A1 (combination of a spectrally dispersive element with monochromatic detection) and A2 (interferometric spectroscopy) are, in themselves, likewise incapable of solving the problem of overlapping emission spectra. However, they are suitable for detecting the spectra information at a sample point.
The combination of methods A1 and A2 with a mathematical algorithm for unmixing overlapping spectra is suitable, in principle, for solving the problem described above (Schäfer application, ASI application). Both methods have the disadvantage of low efficiency compared to the invention described hereinafter. In method A1, only a narrow spectral band is detected at the detection time interval. A plurality of successive measurements are needed to detect a spectrum. This reduces the signal-to-noise ratio of the measurement. Further, repeated illumination of the samples with excitation light damages the fluorescence dyes and the samples themselves (e.g., through phototoxic processes).
In interferometric methods (A2), the detection efficiency is reduced to 50% based on theoretical considerations (citation). In order to acquire spectra from the raw data, a Fourier transform is required. For this purpose, the data are typically subjected to a digital Fourier transform (DFT) or, in case of a quantity of 2n data points, to a fast Fourier transform (FFT). The computing time required for these calculations is not negligible.